This invention relates to a multi-color fluorescence detector, more particularly to a gel electrophoretic apparatus for electrophrescing the four constituent bases of DNA labelled with a corresponding number of fluorescent dyes.
Gel electrophoresis is practiced extensively as a technique for determining the base sequences of DNA and other proteins. Conventionally, the sample to be subjected to electrophoresis is labelled with a radioisotope for analysis but this method has had the problem of being painstaking and time-consuming. Furthermore, the use of radioactive substances always calls for utmost safety and management and analysis cannot be performed in areas other than facilities that clear certain regulations. Under the circumstances, a method that uses fluorophores to label the sample and which detects the fluorescence emitted upon irradiation with light is being reviewed.
In this method, fluorophore-labelled DNA fragments are caused to migrate through a gel and a light excitation portion and a photodetector are provided for each electrophoresis track in an area 15-20 cm below the start point of electrophoresis. The DNA fragments are assayed as they pass through the line connecting the light excitation portion and the photodetector. A typical procedure of the method is described below. First, using as a template the DNA chain to be determined for its base sequence, DNAs of various lengths with known terminal base species are replicated by a method involving an enzymatic reaction (the dideoxy method). Then, the replicated DNAs are labelled with a fluorophore. Stated more specifically, there are prepared a group of adenine (A) fragments, a group of cytosine (C) fragments, a group of guanine (G) fragments and a group of thymine (T) fragments, all being labelled with a fluorophore. A mixture of these fragment groups is injected into separate lane grooves in an electrophoretic gel and, thereafter, a voltage is applied at opposite ends of the gel. Since DNA is a chained polymer with negative charges, it will move across the gel at a rate in inverse proportion to its molecular weight. The shorter the DNA chain (the smaller its molecular weight), the faster will it move and vice versa. This is the principle behind the fractionation of DNA by molecular weight.
The fluorescent dyes currently used for DNA labelling in base sequence determination include fluorescein isothiocyanate (FITC), eosin isothiocyanate (EITC), tetramethylrhodamine isothiocyanate (TMRITC) and substituted rhodamine isothiocyanate (XRITC). Instead of labeling the sample with one fluorescent dye, it may be labelled with two fluorescent dyes such that the fluorescence emitting at two different wavelengths is measured and this contributes to a higher throughput in analysis.
Consider, for example, the case of labelling DNA fragments with FITC and XRITC. Upon illumination with laser light, the dyes emit fluorescence at wavelengths of about 520 nm and 604 nm. When two fluorescent dyes are used, the number of samples that can be analyzed per gel electrolyte is of course doubled but, what is more, the base length that can be identified is increased as compared with the case of using only one dye. The number of lanes that are produced from one sample is four and this is invariable whether one or two dyes are used. On the other hand, the number of samples that can be analyzed per gel electrolyte which is eight in the case of using one fluorescent dye is doubled to 16 when two dyes are used. The base length that can be identified is increased from 400 bp to 450 bp.
To further enhance the throughput of analysis, a multi-color DNA sequencer is being developed in which the four DNA bases are labelled with four fluorescent dyes that emit at different wavelengths. In one type of the multi-color DNA sequencer, a plurality of bandpass filters on a rotating disk are alternately inserted into the optical path of detecting light so as to detect the fluorescence issuing from the sample. However, the need to switch between filters at given time intervals makes it impossible to perform real-time detection. Furthermore, the installation of the rotating mechanism increases not only the complexity but also the overall size of the analyzer. In another type of the multi-color DNA sequencer, a dispersing prism is inserted into the fluorescence detecting optics to separate the light from the sample into different wavelength components. The light from the sample is collected with a condenser lens, made parallel with a collimator and separated into spectral components which are respectively condensed on associated sensors. However, this approach involves mechanistic difficulties due to the need for correcting aberrations such as chromatic aberration and the prism-induced aberration.